This project determines the structure of neuronal and glial cytoplasm, particularly as it pertains to axoplasmic transport. A protein translocator, kinesin, is responsible for the anterograde organelle movements along microtubules, which are the basis of anterograde fast axonal transport. A high molecular weight protein in squid axoplasm, which we have characterized as a cytoplasmic dynein, transports exclusively in the retrograde direction. The functions of this transport system in vivo is also under investigation. Each organelle contacts several microtubules in the axon, so it is the continuous microtubule bundles which constitute the transport pathways down the axon. Much of the pool of kinesin and dynein in vitro is in a soluble form and new immunocytochemical methods had to be developed to determine their distributions in the cytoplasm in relation to the transport pathways. This has now been successful and used to show that kinesin is concentrated on the surfaces of organelles and that there is a cytoplasmic pool of free kinesin, suggesting that there may be two different pools of kinesin with different functions, such as promoting interaction of microtubules with the endoplasmic reticulum or with other microtubules. These interactions are likely to be of fundamental importance in the growth, regeneration, and maintenance of axons.